PEG-6000 induced precipitation of proteins from cold-acclimated Tenebrio molitor larvae

Spectra of PEG-6000-precipitated proteins from cold-acclimated and non-acclimated Tenebrio molitor larvae were comparatively studied. The composition of precipitated proteins from cold-acclimated and non-acclimated T. molitor larvae was shown as differed qualitatively. In particular, the protein spectra of filtrates from cold-acclimated larvae had proteins with molecular weight of 11 kDa, which were absent in non-acclimated insects. There was observed a significant difference in precipitate content at polyethyleneglycol concentrations of 12% and higher during protein precipitation from homogenates of cold-acclimated and non-acclimated larvae. Proteins from the filtrates of cold-acclimated larvae in the presence of 6% PEG-6000 were precipitated by 44 and 56% greater, than those from non-acclimated species at 22 and 4°C, respectively. The increasing of PEG-6000 concentration from 4 to 6% resulted in appearance in the precipitates from cold-acclimated larvae of proteins with molecular-weight range of 30–42.5 kDa, which were absent in non-acclimated insects.Probl Cryobiol Cryomed 2015; 25(4):350-358.

Раніше нами було показано, що в процесі холодової аклімації склад синтезованих білків Тenebrio The feature of protein spatial function is the ability of polypeptide chain to create the certain structure with necessary dynamic properties for implementing biological functions. When discussing the problem of protein stability a special attention is paid to hydrophobic interactions, playing a significant role in macromolecule formation and being one of the important factors of their stabilisation.

Materials and methods
The research was carried-out in T. molitor larvae (Coleoptera: Tenebrionidae) of last age, which namely at this ontogenesis stage became cold-resistant [20]. Larvae were maintained for 3 weeks at 5...7°C for cold-acclimation.
Homogenate was obtained from T. molitor in 0.6% NaCl solution with Na-phosphate buffer 0.1 M (pH 7.4) in amount of 6 larvae per 2 ml buffer. Thereafter it was centrifuged for 10 min at 1800g. The aliquot was collected from the supernatant for electrophoresis and the supernatant was passed through a filter cartridge Sartopore GF2 (Sartorius, Germany) to remove cell detritus. The aliquot was collected from filtrate for electrophoretic study. Filtrate was supplemented with PEG-6000 in 1:1 ratio, stirred and the reaction of precipitation was performed with final PEG concentrations in solution of 2, 4, 6 and 8% at 22 and 4°C for 1.5 hrs. The obtained mixture was centrifuged at 1800g for 10 min and a supernatant was collected into separate vials.
removal. At the second stage we transferred tubing into the PEG-40000-saturated solution and performed concentrating dialysis at 22°C and constant stirring for 90 min. After two-stage dialysis the aliquots were collected for electrophoresis. At each stage of the experiment we measured a protein content according Bradford [18]. The SDS-electrophoresis was carriedout in gradient (10-25%) polyacrylamide gel (PAAG) by the standard technique [13]. For electrophoresis we used the Sigma (USA) and Serva (Germany) reagents. The revealed protein fractions in SDS-PAGE tracks were qualitatively assessed using ImageJ software (National Institutes of Health, USA). Experimental data were statistically processed with the Statgraphics Win 2.1 software using nonparametric Mann-Whitney U-test. Differences at p<0.05 were considered as significant.

Results and discussion
The distribution of hydrophilic and hydrophobic amino acid residues on protein surface plays an important role in its dissolving in polar systems. Right the amount of polar amino acid residues affects the degree of protein hydration.
The high molecular polyethylene glycols are used for precipitation of macromolecules, particularly proteins [7,10] and nucleic acids [14,15,21]. The mechanism of protein precipitation with PEG solutions is based on dehydration. The interaction of PEG with proteins in solution consists in a steric exclusion of PEG molecules from a protein environment due to water binding by polymer [5,19]. As a result the solubility of proteins decreases, and their non-specific aggregation and precipitation occurs. The degree of protein precipitation depends on many parameters, one of which is PEG concentration. L.B. Korolevskaya et al. [9] studied the PEG effect on immune complexes, and demonstrated that at low concentrations of PEG the precipitation of mainly macromolecular immune proteins occurred, while when increasing the polymer concentration the protein complexes of both large and small sizes were precipitated. In particular, when obtaining immunoglobulins PEG was used to assess an aggregate state of the obtained complexes [9]. The use of two PEG concentrations (3 and 4%) was established to differentiate the immune proteins (Ig M, G and A) by size.
be an indirect method for revealing these transformations. The assumption as to the changes in proteins spectrum or their conformation during cold-acclimation has been confirmed by our findings. Fig. 1 and 2 show that as a result of precipitation by means of PEG-6000 within a concentration range of 4-20% the proteins, derived from cold-acclimated T. molitor larvae, precipitate much greater than those from non-acclimated insects.
An increase in PEG-6000 concentration from 4 to 20% led to the augmentation of protein amount in sediment from homogenates of cold-acclimated and non-acclimated T. molitor larvae. A significant difference in the amount of precipitate, obtained from coldacclimated and non-acclimated species, was observed at PEG concentrations of 12% and more (Fig. 1, 2). It should be noted that the temperature, at which the proteins were precipitated, did not significantly affect the amount of precipitated protein. The amount of precipitated protein at 4 and 22°C did not significantly differ in cold-acclimated and non-acclimated larvae.
Since the dissolving the proteins in water is due to their hydration, and the stabilisation of protein molecule depends on its charge and hydration shells, consisting of the oriented water molecules, the effect of any factor, breaking hydration processes and destroying hydration shells of proteins (the main principle of dehydrating agent action), leads to their precipitation.
Therefore we can assume that cold adaptation of the T. molitor larvae is accompanied with conformational changes of proteins, and thus to redistribution of polar and non-polar groups on their surfaces, which contact with solvent, thereby stipulating a decrease in their stability in PEG-6000 solution.
Comparing the protein spectra of filtrates from coldacclimated and non-acclimated T. molitor larvae revealed the qualitative changes, especially notice-able within the molecular weight ranges of 10-15 and 15-85 kDa. In particular, in the MW area of 10-15 kDa, the samples from cold-acclimated T. molitor showed a new low-molecular protein band with 11 kDa MW, and in 15-85 kDa weight area of cold-acclimated insects the proteins with 22.5, 49 and 75 kDa MW disappeared (Fig. 3). We can assume that low molecular proteins (11 kDa) belonged to antifreeze protein family, which are accumulated in T. molitor during cold acclimation [11,12,17].
L.B. Korolevskaya et al. [9] described the aggregation of immunoglobulins in the presence of PEG-6000, moreover the higher was the polymer concentration the bigger was size of aggregates. Since the dimensions of aggregate complexes may affect the electrophoresis when using ionic detergent SDS, and aqueous PEG solutions with molecular weight above 3000 Da have a high viscosity, resulting in distortion of electrophoretic mobility of protein molecules in polymer presence, we selected only low concentrated PEG-6000 for SDS-electrophoretic analysis of protein spectra.
The protein range in precipitates from homogenate filtrates of cold-acclimated and non-acclimated T. molitor larvae in the presence of 4% PEG-6000 at 22°C, was qualitatively different. In the precipitates from cold-acclimated insects no bands with 73 and 18 kDa MW were found, unlike non-acclimated species, where these as well as the proteins with 41.5, 31.5, 28.5, 27.5, 25, 20 and 14.5 kDa MW were present. The composition of protein sediments after precipitating proteins from cold-acclimated and non-acclimated larvae using 4% PEG-6000 solution at 4°C was also different: 45 kD band disappeared in cold-acclimated insects, and the amount of protein of 56 kD MW decreased in respect to the protein spectra from nonacclimated insects. Moreover, in the precipitate from cold-acclimated larvae we observed the proteins with 42.5, 31.5, 30, 20 and 15 kDa MW. In those from acclimated larvae the amount of proteins with 62.5 and 59 kDa MW also increased during precipitation at 4°C (Fig. 4).
Thus, the spectra of revealed protein fractions from filtrates of cold-acclimated and non-acclimated T. molitor larvae, precipitated with 4% PEG-6000 at different temperature conditions were different.

Conclusions
In our findings we revealed the difference in electrophoretic mobility of proteins from filtrates of cold-acclimated and non-acclimated T. molitor larvae. It was proven that the proteins from cold-acclimated T. molitor larvae precipitated in greater amount, than those from non-acclimated insects, that might indicate conformational changes in proteins during cold adaptation.